PMCA for High-Throughput Detection of Prions using the UIP400MTP Sonicator

The UIP400MTP multi-well plate sonicator offers a powerful solution for high-throughput sample preparation in protein misfolding cyclic amplification (PMCA). By delivering uniform energy distribution across multiple wells and maintaining precise sonication parameters, this system enables the simultaneous processing of numerous samples with exceptional reproducibility. These capabilities are critical for optimizing PMCA to detect prions at low concentrations in challenging biological samples, such as saliva, where assay inhibitors can obscure results.

Prion diseases, such as chronic wasting disease (CWD) in cervids and Creutzfeldt-Jakob disease (CJD) in humans, are neurodegenerative disorders caused by misfolded prion proteins (PrP^Sc). These diseases often involve low levels of infectious prions in bodily fluids, such as saliva, blood, and urine, complicating diagnosis and research. Horizontal transmission of CWD through prions shed in environmental matrices has particularly significant implications for wildlife management and ecological health. Similarly, in diseases like Creutzfeldt-Jakob disease, reliable amplification of misfolded prion proteins from human samples is crucial for advancing diagnostics and understanding disease progression.

Applying a modified PMCA protocol allows to bypass common assay inhibitors (e.g., mucins in saliva) and achieve high sensitivity in detecting prions that would be otherwise undetectable or ambiguous using techniques such as real-time quaking-induced conversion (RT-QuIC). These advancements enhance prion detection for various diseases, making PMCA an indispensable tool for prion research and diagnostics. The multi-well plate sonicator UIP400MTP facilitates high-throughput PMCA sonicating numerous samples in microplates (e.g. 6-, 24-, or 96-well plates) or vials in a tube rack.

Protocol for High-Throughput Protein Misfolding Cyclic Amplification (PMCA)

The following protocol allows the efficient processing of high sample number under the exactly same conditions for robust research results.

Sample Preparation

Starting Material:
Prepare samples by:

• Resuspending sarkosyl extraction pellets in PMCA substrate.
• Directly spiking brain homogenates or blood samples with prion seeds.

Substrate:

• Use a 10% (wt/vol) brain homogenate prepared from transgenic mice over-expressing PrP^C (e.g., Tg mice).
• Homogenize the brain tissue in:
  – 1× PBS.
  – 150 mM NaCl.
  – 1% Triton X-100.
• Store the substrate at -80ºC until use.

Sample Setup in Microplate or Tubes:
Tubes:

• Add 90 μL of brain homogenate substrate and seed with 10 μL of sample (e.g., blood, brain homogenate, or sarkosyl pellet).
• Place 3 Teflon beads (1.59-mm or 2.38-mm diameter) in each 0.2 mL tube.
• Mount the tubes in a rack compatible with the UIP400MTP sonicator.

6-Well Microplate:

• Add 5 mL of brain homogenate substrate and seed with 500 μL of sample per well.
• Add 3 Teflon beads to each well.

PMCA Procedure

Placement:
Place the tube rack or 6-well microplate into the UIP400MTP sonicator according to the manufacturer’s instructions.

Cycling Program:
Perform 144 PMCA cycles as follows:

• Incubation: 29 minutes and 30 seconds at 37°C.
• Sonication: 30 seconds at 60% amplitude.
• Monitor the temperature: Use the pluggable temperature sensor to monitor sample temperature and program the UIP400MTP to a max. temperature of 48–50°C.

Subsequent Rounds:
After completing the first round of 144 cycles, transfer an aliquot of the amplified material:

• Dilute 10-fold into fresh transgenic mouse brain homogenate substrate.
• Perform 96 PMCA cycles for subsequent rounds, maintaining the same sonication parameters.
• Continue for the desired number of rounds (typically up to 5).

Detection of PrP^Sc

1. Proteinase K Digestion:
   – Treat samples with Proteinase K (50 μg/mL) at 37°C for 1 hour.
   – Terminate digestion by adding SDS-sample buffer and boiling for 10 minutes.
2. Western Blot Analysis:
   – Analyze digested samples using:
   – 6H4 or PRC1 anti-PrP antibodies.
   – Perform SDS-PAGE and transfer to PVDF membranes for detection.

 

 

Process More Samples for More Robust Results

The UIP400MTP multi-well plate sonicator significantly enhances the efficiency and scalability of protein misfolding cyclic amplification (PMCA), addressing the traditionally time-consuming nature of the procedure. By allowing the simultaneous processing of up to 96 samples in a 96-well plate, the system streamlines PMCA workflows while maintaining precise and uniform sonication conditions across all wells. This high-throughput capability minimizes manual handling, reduces labor-intensive steps, and ensures reproducibility, making it an indispensable tool for prion research. Whether investigating chronic wasting disease or Creutzfeldt-Jakob disease, the UIP400MTP facilitates large-scale studies with greater efficiency, enabling researchers to meet the demands of modern diagnostic and scientific applications.

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Literature / References

• FactSheet UIP400MTP Multi-well Plate Sonicator – Non-Contact Sonicator – Hielscher Ultrasonics
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• Mochizuki, Chika; Taketomi, Yoshitaka; Irie, Atsushi; Kano, Kuniyuki; Nagasaki, Yuki; Miki, Yoshimi; Ono, Takashi; Nishito, Yasumasa; Nakajima, Takahiro; Tomabechi, Yuri; Hanada, Kazuharu; Shirouzu, Mikako; Watanabe, Takashi; Hata, Kousuke; Izumi, Yoshihiro; Bamba, Takeshi; Chun, Jerold; Kudo, Kai; Kotani, Ai; Murakami, Makoto (2024): Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation. BioRxiv 2024.
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